Does Antibody Avidity to Plasmodium falciparum Merozoite Antigens Increase with Age in Individuals Living in Malaria-Endemic Areas?

High-avidity antibodies (Abs) are acquired after a few Plasmodium falciparum infections in low transmission areas, but it remains unclear if Ab avidity to different merozoite antigens increases with age in individuals with persistent antigenemia and, if so, when a fully mature Ab response occurs. The study used plasma samples collected between 1996 and 1998 from 566 individuals aged 4 to 84 years in Simbok, Cameroon, where residents received an estimated 1.6 infectious mosquito bites/person/night. Plasma samples were examined for Ab levels (median fluorescence intensity [MFI]) and Ab avidity index (AI) (where AI = [MFI after treatment with 2 M NH4SCN/MFI without salt] × 100) using a bead-based multiplex immunoassay for recombinant AMA1, EBA-175, MSP1-42 (3D7, FVO), MSP2 (3D7, Fc27), and MSP3. Blood-smear positivity for P. falciparum declined with age from 54.3% at 4 to 5 years to 18% at 16 to 40 years and <11% at >40 years of age, although most individuals had submicroscopic parasitemia. Ab affinity maturation, based on age-related patterns of median AI, percentage of individuals with AI of ≥50, and strength of association between MFI and AI, occurred at different rates among the antigens; they developed rapidly before age 4 years for AMA1, increased gradually with age for EBA-175 and MSP1 until ∼16 to 25 years, but occurred negligibly for MSP2 and MSP3. In a hyperendemic area with perennial transmission, affinity maturation resulting in an increase in the proportion of high-avidity Abs occurred for some merozoite antigens, in parallel with a decline in malaria slide passivity, but not for others.

Feasibility of microbial sample collection on the skin from people in Yaoundé, Cameroon.

Characterization of microbial communities in the skin in healthy individuals and diseased patients holds valuable information for understanding pathogenesis of skin diseases and as a source for developing novel therapies. Notably, resources regarding skin microbiome are limited in developing countries where skin disorders from infectious diseases are extremely common. A simple method for sample collection and processing for skin microbiome studies in such countries is crucial. The aim of this study is to confirm the feasibility of collecting skin microbiota from individuals in Yaoundé, a capital city of Cameroon, and subsequent extraction of bacterial DNA in a resource limited setting. Skin swabs from several individuals in Yaoundé were successfully obtained, and sufficient amount of bacterial 16S ribosomal RNA-coding DNA was collected, which was confirmed by quantitative PCR. The median copy number of 16S ribosomal RNA gene varied across participants and collection sites, with significantly more copies in samples collected from the forehead compared to the left and right forearm, or back. This study demonstrated that collecting surface skin microbes using our swabbing method is feasible in a developing country. We further showed that even with limited resources, we could collect sufficient amount of skin microbiota from the inhabitants in Yaoundé where no studies of skin microbiome were reported, which can be passed to further metagenomic analysis such as next generation sequencing.

PCR-based detection of Plasmodium falciparum in saliva using mitochondrial cox3 and varATS primers.

BACKGROUND: Sampling of saliva for diagnosing Plasmodium falciparum infections is a safe, non-invasive alternative to sampling of blood. However, the use of saliva presents a challenge because lower concentrations of parasite DNA are present in saliva compared to peripheral blood. Therefore, a sensitive method is needed for detection of parasite DNA in saliva. This study utilized two recently reported "ultra-sensitive" PCR assays based on detection of the P. falciparum mitochondrial cox3 gene and the multi-copy nuclear varATS gene. The ultra-sensitive assays have an advantage over standard 18S rRNA gene-based PCR assay as they target genes with higher copy numbers per parasite genome. Stored saliva DNA samples from 60 Cameroonian individuals with infections previously confirmed by 18S rRNA gene PCR in peripheral blood were tested with assays targeting the cox3 and varATS genes. RESULTS: Overall, the standard 18S rRNA gene-based PCR assay detected P. falciparum DNA in 62% of the stored saliva DNA samples, whereas 77 and 68% of the samples were positive with assays that target the cox3 and varATS genes, respectively. Interestingly, the ultra-sensitive assays detected more P. falciparum infections in stored saliva samples than were originally detected by thick-film microscopy (41/60 = 68%). When stratified by number of parasites in the blood, the cox3 assay successfully detected more than 90% of infections using saliva when individuals had > 1000 parasites/µl of peripheral blood, but sensitivity was reduced at submicroscopic parasitemia levels. Bands on electrophoresis gels were distinct for the cox3 assay, whereas faint or non-specific bands were sometimes observed for varATS and 18S rRNA that made interpretation of results difficult. Assays could be completed in 3.5 and 3 h for the cox3 and varATS assays, respectively, whereas the 18S rRNA gene assays required at least 7 h. CONCLUSIONS: This study demonstrates that a PCR assay targeting the cox3 gene detected P. falciparum DNA in more saliva samples than primers for the 18S rRNA gene. Non-invasive collection of saliva in combination with the proposed cox3 primer-based PCR assay could potentially enhance routine testing of P. falciparum during disease surveillance, monitoring, and evaluation of interventions for malaria elimination.

Association of Antibodies to VAR2CSA and Merozoite Antigens with Pregnancy Outcomes in Women Living in Yaoundé, Cameroon.

Plasmodium falciparum infections are serious in pregnant women, because VAR2CSA allows parasitized erythrocytes to sequester in the placenta, causing placental malaria (PM). In areas of endemicity, women have substantial malarial immunity prior to pregnancy, including antibodies to merozoite antigens, but produce antibodies to VAR2CSA only during pregnancy. The current study sought to determine the importance of antibodies to VAR2CSA and merozoite antigens in pregnant women in Yaoundé, Cameroon, where malaria transmission was relatively low. A total of 1,377 archival plasma samples collected at delivery were selected (at a 1:3 ratio of PM-positive [PM+] to PM-negative [PM-] women) and screened for antibodies to full-length VAR2CSA and 7 merozoite antigens. Results showed that many PM+ women and most PM- women lacked antibodies to VAR2CSA at delivery. Among PM+ women, antibodies to VAR2CSA were associated with a reduced risk of having high placental parasitemia (odds ratio [OR], 0.432; confidence interval [CI], 0.272, 0.687; P = 0.0004) and low-birth-weight (LBW) babies (OR = 0.444; CI, 0.247, 0.799; P = 0.0068), even during first pregnancies. Among antibodies to the 7 merozoite antigens, i.e., AMA1, EBA-175, MSP142, MSP2, MSP3, MSP11, and Pf41, only antibodies to MSP3, EBA-175, and Pf41 were associated with reduced risk for high placental parasitemias (P = 0.0389, 0.0291, and 0.0211, respectively) and antibodies to EBA-175 were associated with reduced risk of premature deliveries (P = 0.0211). However, after adjusting for multiple comparisons significance declined. Thus, in PM+ women, antibodies to VAR2CSA were associated with lower placental parasitemias and reduced prevalence of LBW babies in this low-transmission setting.

A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum.

Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation. A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads internalized.

Connections between the medial temporal cortex and the CA1 subfield of the hippocampal formation in the Japanese monkey (Macaca fuscata).

The connections between the medial temporal cortical areas and CA1 of the hippocampus were examined in the Japanese monkey (Macaca fuscata) by means of retrograde and anterograde tract-tracing methods with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) and fluorescent dyes (Fast Blue and Diamidino Yellow). The posterior parahippocampal (areas TF1, TF2, and TH), perirhinal (areas 35 and 36), and ventral inferotemporal areas (areas TEav and TEpv) were reciprocally connected with CA1. Projection fibers from CA1 to the medial temporal cortical areas originated in the pyramidal cell layer, whereas those from the medial temporal cortical areas to CA1 terminated in the molecular layer. Each of these cortical areas was reciprocally connected with the entire rostrocaudal extent of CA1. However, the intensity of the connections varied along the rostrocaudal axis of CA1: areas TH and TF2 were connected most markedly with the anterior and middle parts of CA1, respectively. Areas TF, 35, 36, TEav, and TEpv were connected predominantly with the posterior part of CA1. In the coronal plane of CA1, labeled cells were located in proximal CA1 (i. e., near the prosubiculum), but not in distal CA1 (i.e., near CA2). The medial temporal cortical areas in direct reciprocal connection with CA1 were presumed to be involved in the memory system, especially in the system for declarative memory.

Neural connections of auditory association cortex with the posterior cingulate cortex in the monkey.

Clinical studies have indicated that the posterior cingulate cortex is intimately involved in verbal and auditory memory. The present study was performed to obtain anatomical evidence for the above proposal. The connections of the auditory cortical areas with the posterior cingulate cortex in the macaque monkey were examined by retrograde and anterograde tracing methods using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). WGA-HRP was injected into either area TA, TB or TC in the superior temporal auditory cortex. Area TA was reciprocally connected with the posterior cingulate cortex, whereas areas TC and TB were not. The rostral two-thirds of area TA had major connections with the caudomedial lobule in the retrosplenial cortex (CML of Goldman-Rakic et al., 1984) and a minor one with area 23b. The caudal third of area tA was connected only with area 23b. However, neither labeled cells nor terminals were observed in areas 23a, 23c, 29, 30 or 31 in the posterior cingulate cortex following a WGA-HRP injection into the caudal, intermediate or rostral portion of area TA. The present finding suggests that verbal and auditory memory impairment in patients with damage to the posterior cingulate cortex is largely due to damage to the CML and area 23b and not to the other posterior cingulate areas.

Two-stage model of visual pattern discrimination learning in macaque monkeys (Macaca mulatta and Macaca fuscata).

To investigate the nature underlying the process of pattern discrimination learning, a series of seven experiments on seven working assumptions were undertaken. The main findings are as follows. The pattern discrimination learning consists of two time-dependent stages: The initial or first learning stage is the period of performance at chance, and the succeeding or second stage is the period of performance from just above the chance to a criterion level. The duration of the first stage is dependent on the degrees of cue-response separations, whereas that of the second stage is independent of. During the first stage, monkeys do not attend to the discriminative cue even at small cue-response separations, whereas during the second stage, they achieve pattern discrimination, or pattern perception and cognition, regardless of cue-response separations. After having learned the first pattern task with a cue-response separation, they learned new pattern tasks by means of the second stage, showing marked saving of the duration of the first stage. The findings in the present studies indicate that the first stage of learning is predominantly involved in the process of attending to the discriminative cues remote from the response site (a selective attention to the cue), whereas the second stage is concerned with the process of perception and cognition of the discriminative cue.

A role of amygdala in visual perception and cognition in macaque monkeys (Macaca fuscata and Macaca mulatta).

To elucidate the role of the amygdala in visual perception and cognition, the effects of ablations of the amygdala and inferotemporal cortex on several visual tasks were compared with each other, and also the distribution patterns of the projections between them were investigated. The findings indicate that the inferotemporal cortex plays a critical role in visual perception, cognition and memory, whereas the amygdala is involved fundamentally in controlling emotional and motivational behavior. However, the amygdala is concerned with vision in the following ways: It receives neutral visual information highly processed in the visual cortex, invests the information with emotional and motivational significance through interactions with the cortical and subcortical systems of emotion and motivation, and then it returns the information coded to the visual areas to be re-processed; to be consciously perceived in area TEO, and to be meaningfully cognized, recognized and memorized in areas TE and TEG. Therefore, two channel model regarding the mechanism of visual information processing in the inferotemporal cortex is proposed: A first channel is concerned with processing neutral information, while the second one, with processing meaning information coded emotionally and motivationally in the amygdala. In addition, the present studies demonstrate that area TEG, which is cytoarchitecturally a transitional area between areas TE and TG and whose functional significance has remained unclear, is involved significantly in visual cognition rather than visual perception.

Direct projections from the ventral TE area of the inferotemporal cortex to hippocampal field CA1 in the monkey.

Anterogradely labeled terminals were found densely in field CA1, but not in any other field, of the hippocampal formation following injections of horseradish peroxidase into the ventral TE area of the inferotemporal cortex, whereas no label was seen in any field of the hippocampal formation following injections into the dorsal TE area. The labeled terminals were distributed mainly in a medial part of the stratum moleculare of field CA1 throughout its rostrocaudal extent. The present finding provides the first demonstration of direct projections from the inferotemporal cortex to the hippocampal formation.

A direct projection from hippocampal field CA1 to ventral area TE of inferotemporal cortex in the monkey.

Retrogradely labeled cells were found in field CA1 of the hippocampal formation in monkeys following injections of horseradish peroxidase in ventral area TE of inferotemporal cortex. No hippocampal label was found following injections in dorsal area TE and area TEO. The findings provide the first demonstration of a direct projection from hippocampal field CA1 to area TE.

Amygdalar connections with middle and inferior temporal gyri of the monkey.

The origins and terminations of the amygdalar connections with middle (ITm) and inferior temporal (ITi) gyri of inferotemporal cortex (area TE) were studied in Japanese monkeys by the horseradish peroxidase method. The ITm gyrus received a major projection from the lateral basal nucleus and a minor one from the accessory basal nucleus of the amygdala, whereas it sent a major projection to the lateral nucleus and a minor one to the lateral basal nucleus. The ITi gyrus had only minor amygdalar projections from the medial basal nucleus and to the medial basal and lateral nuclei.

Amygdalofugal and amygdalopetal connections with modality-specific visual cortical areas in macaques (Macaca fuscata, M. mulatta, and M. fascicularis).

The origins and terminations of the amygdaloid connections with the modality-specific visual cortical areas TEa (anterior TE area), TEp (posterior TE area), TEO, V4, V2, MST (medial superior temporal visual area), MT (middle temporal visual area), and V1 were studied in macaques. These were compared with the amygdaloid connections of a vision-related polysensory area TG by making cortical injections of horseradish peroxidase (HRP) and incubating the sections with tetramethylbenzidine (TMB) as the chromogen. Both areas TEa and TEp receive a major projection from the lateral basal nucleus and a minor one from the accessory basal nucleus of the amygdaloid complex, whereas these areas send a major projection to the lateral nucleus and a minor one to the lateral basal nucleus. Areas TEO, V4, V2, MST, MT, and V1 receive projections only from the lateral basal nucleus; none of them project to any amygdaloid nucleus. Thus, the amygdalofugal projections are more widespread and more complex than the amygdalopetal projections. These findings indicate that the connections between the amygdaloid nuclei and the visual areas are generally nonreciprocal and underlie the importance of a feedback mechanism from the amygdala to the visual cortical areas in visual information processing. There appears to be a caudorostral (occipitotemporal) gradient in the distribution and density of the amygdaloid projections, which become progressively more widespread and heavier among the progressively more rostral visual areas (from area V1 to area TEa). The amygdaloid connections with area TG are distinctly different from the connections with the visual areas. Area TG is reciprocally connected mainly with the periamygdaloid cortex, and with the lateral, accessory basal, and medial basal nuclei of the amygdala as well.

Analysis of local and wide-field movements in the superior temporal visual areas of the macaque monkey.

The middle temporal (MT) and medial superior temporal (MST) areas of the macaque cortex have many cells that respond to straight movements in the frontoparallel plane with directional selectivity (D cells). We examined their responses to movements of a bar, of a wide dot pattern, and to combined movements of the two in anesthetized and immobilized animals. D cells in MT showed a wide variety in the strength of the inhibitory field surrounding the excitatory center field. Responses of SI+-type cells to a bar moving across the excitatory field were suppressed when a wide dot pattern moved over the surround field in the same direction and at the same speed as the bar. Inhibition was selective to the direction and speed of the surround movement, and the effective area for inhibition occupied a wide area, which expanded in all radial directions. Responses of SI- -type cells to a center bar movement were changed little by a conjoint movement over the surround field. Consequently, SI- -type cells responded to wide-field movement as well as to stimuli confined within the excitatory field. Although D cells in MST commonly had large excitatory fields, a proportion of them (Figure type) responded to bar movement much more strongly than to wide-field movement. Their responses to a bar movement were suppressed direction-selectively by a conjoint movement of a wide dot-pattern background. The effective area for inhibition coexisted with the excitatory field in these cells. MST cells of the Nonselective type responded comparably well to the two stimuli, and those of the Field type responded much more strongly to wide-field movement than to bar movement. It is thus suggested that MT cells of the SI+ type and MST cells of the Figure type can detect a difference between movements of an object and its wide background, whereas MST cells of the Field type can detect a conjoint movement of a wide field, neglecting the movements of a single object.

Integration of direction signals of image motion in the superior temporal sulcus of the macaque monkey.

Using anesthetized and paralyzed monkeys, we have studied the visual response properties of neurons in the cortical area surrounding the middle temporal area (MT) in the superior temporal sulcus (STS). Systematic electrode penetrations revealed that there is a functionally distinct region where three classes of directionally selective cells with large receptive fields cluster. This region is anteriorly adjoined to the dorsal two-thirds of MT, has a width of 4-5 mm mediolaterally, and therefore may correspond to the dorsal part of the medial superior temporal area (MST), which was previously defined as a MT-recipient zone. One class of cells responded to a straight movement of patterns in the frontoparallel plane with directional selectivity (D cells: 217/422, 51.4%). The second class of cells selectively responded to an expanding or contracting size change of patterns (S cells: 66/422, 15.7%). These cells responded neither to a change in width of a slit of any orientation or any length, nor to a change in brightness. The third class of cells responded only to a rotation of patterns in one direction (R cells: 58/422, 13.7%). A majority of these cells (41/58) responded to the clockwise or counterclockwise rotation of patterns in the frontoparallel plane (Rf cells), while the rest responded to a rotation of patterns in depth (Rd cells). We will suggest that these cells acquire the ability to discover whole events of visual motion--i.e., unidirectional straight movement, size change (radial movement), and rotation--by integrating elemental motion information extracted by MT cells. The receptive fields of D, S, and Rf cells can be constructed by converging signals of MT cells, the preferred directions of which are arranged in parallel (D cells), radially (S cells), and circularly (Rf cells). The receptive fields of Rd cells can be constructed, in turn, by the convergence of signals of S cells.

Laminar origin of direct projection from cortex area V1 to V4 in the rhesus monkey.

Direct projection from cortex area V1 to V4 in the rhesus monkey was demonstrated by means of retrograde axonal transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). This projection originated at the layers 2 and 3 of the only representation area of central visual field (0-6 degrees) in V1 and terminated at the central representation of V4, whereas there was no projection between the extracentral representations of V1 and V4. Correlation of the present finding with the previous findings suggests that this projection is predominantly involved in the system of color information processing.

Direct projection from the dorsal lateral geniculate nucleus to the prestriate cortex in macaque monkeys.

The enzyme horseradish peroxidase (HRP) was separately injected into striate, prestriate, inferotemporal, and parietal cortices in 19 macaque monkeys, and the lateral geniculate nucleus (LGN) was examined for retrograde transport. Labeled LGN cells were identified only in the animals, with HRP injections into the striate and prestriate cortex. Following injections into either of these regions, labeled cells were found in both parvocellular and magnocellular regions of the ipsilateral LGN only, in keeping with the topographic relation of HRP injection sites in the cortex to labeled areas in the LGN. It was also found that (1) labeled LGN cells were less numerous in both laminar and interlaminar zones following HRP injection into the prestriate cortex, whereas following HRP injection into the striate cortex labeled cells were found almost exclusively in the laminae, and localized to a wedge-shaped region; (2) following HRP injection into the prestriate cortex, the mean sizes of the labeled parvocellular and magnocellular cells, estimated in projected diameter, were almost the same, these means being significantly larger than the mean size of labeled parvocellular cells and much smaller than that of labeled magnocellular cells following HRP injection into the striate cortex; (3) the shapes of the labeled LGN cells following HRP injection into the prestriate cortex were ovoid, fusiform, or triangular (or multipolar), whereas those following HRP injection into the striate cortex were uniformly ovoid or round. The above findings following HRP injections into the prestriate cortex in normal monkeys were confirmed by HRP injections into the prestriate cortex of monkeys whose striate cortex had been removed several months prior to the injection; labeled cells were found in confines of areas of retrograde degeneration in the LGN and their labeling pattern was the same as that in intact animals. It was concluded that in macaque monkeys, just as in the cat, a geniculoprestriate projection system exists; it was suggested that there are two parallel system of visual information processing from the LGN to the prestriate cortex, a direct one and in indirect one through the striate cortex.